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Journal of Biochemistry 2007 142(1):55-64; doi:10.1093/jb/mvm107
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© 2007 The Japanese Biochemical Society.

A Cholesterol-independent Membrane Microdomain Serves as a Functional Counter-receptor for E-selectin at the Colo201 Cell Surface and Initiates Signalling on E-selectin Binding

Chikako Suzuki and Naoya Kojima*

The Institute of Glycotechnology, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan

*To whom correspondence should be addressed. Tel: 0463-58-1211 (Ex. 4645), Fax: 0463-50-2012, E-mail: naoyaki{at}keyaki.cc.u-tokai.ac.jp

Received March 25, 2007; Accepted April 20, 2007


  Abstract

The present study demonstrates that the functional counter-receptors for E-selectin at the cell surface of Colo201 human colon cancer cells are localized in detergent-insoluble membrane microdomains (DIM). Following isolation of counter-receptors from whole cell lysates using E-selectin-coupled magnetic beads followed by sucrose density gradient separation, both sialyl Lewis a (SLea)- and sialyl Lewis x (SLex)-carrying glycoproteins which had bound to the E-selectin-beads were distributed in detergent-soluble fractions as well as DIM. In contrast, following isolation of counter-receptors directly from the cell surface, SLea-carrying glycoproteins which had bound to E-selectin-beads at the cell surface were localized only in DIM, together with a Src family kinase, Lyn, while SLex-carrying glycoproteins were not detected in any fraction. The counter-receptors were distributed in a diffuse pattern on the cell surface but clustered following E-selectin binding, leading to the subsequent phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of the cells with methyl-ß-cyclodextrin, a cholesterol-depleting drug, had little effect on either the association of SLea-carrying glycoproteins and Lyn with the domain or ERK phosphorylation. Thus, the functional counter-receptors and Lyn are co-localized in a cholesterol-independent microdomain and create a physiological domain (‘glycosynapse’) at the cell surface that initiates signalling in cancer cells upon binding to E-selectin.

Key Words: colon cancer cell, detergent-insoluble microdomain, E-selectin, glycosynapse, sialyl Lewis a, Src family kinase

Abbreviations: DIM, detergent-insoluble membrane microdomain; FITC, fluoroscein isothiocyanate; MCD, methyl-ß-cyclodextrin; Slea, sialyl Lewis a; Slex, sialyl Lewis x


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